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Advances in Geometric Techniques for Analyzing Blebbing in Chemotaxing Dictyostelium Cells

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Advances in Geometric Techniques for Analyzing Blebbing in Chemotaxing Dictyostelium Cells City University of New York (CUNY) CUNY Academic Works Publications and Research Hunter College 2019 Advances in geometric techniques for analyzing blebbing in chemotaxing Dictyostelium cells Zully Santiago CUNY Hunter College John Loustau CUNY Hunter College David Meretzky CUNY Hunter College Devarshi Rawal CUNY Hunter College Derrick Brazill CUNY Hunter College How does access to this work benefit ou?y Let us know! More information about this work at: https://academicworks.cuny.edu/hc_pubs/559 Discover additional works at: https://academicworks.cuny.edu This work is made publicly available by the City University of New York (CUNY). Contact: [email protected] RESEARCH ARTICLE Advances in geometric techniques for analyzing blebbing in chemotaxing Dictyostelium cells 1 2 2 2 3 Zully Santiago , John Loustau *, David Meretzky , Devarshi Rawal , Derrick BrazillID * 1 Department of Biological Sciences, Hunter College and the PhD Program in Biology, Graduate Center, CUNY, New York, NY United States of America, 2 Department of Mathematics and Statistics, Hunter College, CUNY, New York, NY, United States of America, 3 Department of Biological Sciences, Hunter College and the PhD Programs in Biology and Biochemistry, Graduate Center, CUNY, New York, NY United States of a1111111111 America a1111111111 a1111111111 * [email protected] (JL); [email protected] (DB) a1111111111 a1111111111 Abstract We present a technical platform that allows us to monitor and measure cortex and mem- brane dynamics during bleb-based chemotaxis. Using D. discoideum cells expressing OPEN ACCESS LifeAct-GFP and crawling under agarose containing RITC-dextran, we were able to simulta- Citation: Santiago Z, Loustau J, Meretzky D, Rawal neously visualize the actin cortex and the cell membrane throughout bleb formation. Using D, Brazill D (2019) Advances in geometric techniques for analyzing blebbing in chemotaxing these images, we then applied edge detect to generate points on the cell boundary with Dictyostelium cells. PLoS ONE 14(2): e0211975. coordinates in a coordinate plane. Then we fitted these points to a curve with known x and y https://doi.org/10.1371/journal.pone.0211975 coordinate functions. The result was to parameterize the cell outline. With the parameteriza- Editor: Michael Koval, Emory University School of tion, we demonstrate how to compute data for geometric features such as cell area, bleb Medicine, UNITED STATES area and edge curvature. This allows us to collect vital data for the analysis of blebbing. Received: October 26, 2018 Accepted: November 14, 2018 Published: February 14, 2019 Copyright: © 2019 Santiago et al. This is an open access article distributed under the terms of the 1 Introduction Creative Commons Attribution License, which permits unrestricted use, distribution, and Cells must modify their motile behavior when encountering varying conditions. They must reproduction in any medium, provided the original travel through multiple environments as they participate in a variety of biological phenomena author and source are credited. including foraging for food, embryogenesis, development, wound healing, immune response, Data Availability Statement: All relevant data are and cancer metastasis. There are two distinct modes of motility cells utilize depending on their within the paper and its Supporting Information environment [1], [2]. When crawling on top of a substrate with limited resistance to move- files. ment, a two dimensional environment, cells use filopodia, lamellipodia, or pseudopodia as Funding: This work was supported by National their main mode(s) of motility where actin is continuously cycled to the front of the cell, push- Institutes of Health - National Institute on Minority ing the cell's membrane forward in the direction of movement. When crawling through a sub- Health and Health Disparities, 8 G12 MD007599 strate or between cells where resistance is higher, a three dimensional environment, cells use (https://www.nimhd.nih.gov/); National Science blebs as their main mode of motility. During bleb-based motility, the front of the cell makes a Foundation, Division of Molecular and Cellular series of blister-like protrusions in the direction of movement where the cell's membrane Biosciences, MCB-1244162 (https://www.nsf.gov/ div/index.jsp?div=MCB); The Professional Staff detaches from the actin cortex [3]. This is driven in part by the increased intracellular pressure Congress-City University of New York, 69271 00 associated with moving through a three dimensional environment. A variety of cell types have 47 (https://www.rfcuny.org/RFWebsite/research/ been shown to utilize bleb-based motility in three dimensional environments: skeletal muscle PLOS ONE | https://doi.org/10.1371/journal.pone.0211975 February 14, 2019 1 / 21 Advances in geometric techniques for analyzing blebbing in chemotaxing Dictyostelium cells content.aspx?catID=1190); and National Institutes stem cells, zebrafish primordial germ cells, cancer cells, Entamoeba histolytica and Dictyoste- of Health - National Institute of General Medical lium discoideum [4], [5], [6], [7], [8], [9] and [10]. Sciences (RISE), GM060665 (https://www.nigms. The formation of a bleb follows three general steps with distinct membrane and cortex nih.gov/Training/RISE). The funders had no role in study design, data collection and analysis, decision characteristics (Fig 1): 1) the membrane detaches from the cortex, making a blister-like protru- to publish, or preparation of the manuscript. sion at the cell front; 2) the new cortex begins forming at the new position of the membrane while the original cortex behind the detachment begins to disassemble; and 3) the original cor- Competing interests: The authors have declared that no competing interests exist. tex vanishes where the new cortex is fully assembled and associated with the membrane. In recent years, several researchers have studied bleb formation from different points of view. In [11], Charras et al. collected biophysical data from the cell during blebbing, leading them to conclude that the blebbing process is the result of pressure changes. They proposed a model for bleb nucleation. However, these conditions were necessary but not sufficient. Using classical geometric constructs, Wooley et al. [12] modelled bleb development and were able to explain several bleb shapes that occur in nature. Guy and Strychalsky [13], [14] considered the same process from the point of view of computational fluid dynamics. By simulating the fluid flows, they were able to include secondary blebbing in their model. Zatulovskiy, Tyson, Bretschneider, and Kay [3] visualized the cortex and membrane dynamics during bleb forma- tion in D. discoideum using an under agarose assay and introduced differential geometry via membrane/cortex curvature to the process. They showed that curvature does play a role as bleb location is biased toward areas of negative curvature. However, it is apparent from this work that there are other factors at play. Collier et al. [15] proposed that cell surface energy may help predict bleb nucleation sites, and that membrane curvature is only one factor in the cell surface energy calculations. This is in keeping with [16], where the authors correlate the presence of Myosin-II with bleb formation. There are several proposed mechanisms necessary for bleb formation [17]. However, these do not fully explain directional bleb-based motility. Blebs appear as a consequence of the three dimensional environmental force transferred to increased hydrostatic force on the cortex/ membrane complex. The central question is why the blebs appear on the anterior face leading Fig 1. Bleb formation can be identified by cortex-to-membrane positioning. At T1, the membrane detaches from the cortex, initiating a bleb. At T2, an actin scar in the original location of the cortex disassembles as the cortex begins to reform at the new location of the membrane. At T3, the cortex is bound to the membrane in the bleb. https://doi.org/10.1371/journal.pone.0211975.g001 PLOS ONE | https://doi.org/10.1371/journal.pone.0211975 February 14, 2019 2 / 21 Advances in geometric techniques for analyzing blebbing in chemotaxing Dictyostelium cells to coordinated cell movement. Even if blebs tend to occur at sites of negative curvature [18] and [3] or high surface energy [15], these do not explain why blebs are more likely on the ante- rior face. Indeed, there are negative curvature segments and high energy locations throughout the cell boundary. In order to collect the data needed to address this conundrum, large num- bers of blebbing cells need to be imaged at high enough resolution to visualize cortex and membrane structures. In addition, these images need to be analyzed to accurately measure and quantify membrane curvature and bleb size throughout bleb-based motility. In this letter, we describe the technical procedures permitting us to collect images of the cortex and membrane during blebbing as well as the automated computer application that approximates the cell boundary with a B-spline and measures curvature, allowing for a high throughput geometric analysis of blebbing during chemotaxis. This letter is organized as fol- lows. In the Materials and Methods, we state in turn, cell preparation, microscopy procedures, edge detection and geometric modeling functions. In the Results, we apply the methods to D. discoideum. In particular, we produce a parametrized representation of the cell boundary. We then proceed to show some applications of our
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